JP2022058345A - Chimeric antigen receptors targeting b-cell maturation antigen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide: compositions that can be used in methods of treating multiple myeloma; and the methods.

SOLUTION: The invention provides an isolated and purified nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprising an antigen recognition moiety and a T-cell activation moiety, where the antigen recognition moiety is directed against the B-cell maturation antigen (BCMA). The invention also provides host cells, such as T-cells or natural killer (NK) cells, expressing the CAR.

SELECTED DRAWING: None

COPYRIGHT: (C)2022,JPO&INPIT

Description

[Cross-reference with related applications]
This patent application claims the benefit of US Provisional Application No. 61 / 622,600 (Filing Date: April 11, 2012).
The entire application is included in the present application by reference.

[Incorporation into the present application by referring to the property submitted electronically]
The readable nucleic acid / amino acid sequence listings by the following computers submitted at the same time as this application are incorporated herein by reference in their entirety:
A 42,589 byte ASCII (text) file named "712361 # ST25.TXT," (created March 14, 2013).

[Background of invention]
Multiple myeloma (MM) is a malignant tumor characterized by the accumulation of clonal plasma cells (for example, Palumbo et al., New England J. Med., 364 (11): 1046-1060 (2011). ), And Lonial et al., Clinical Cancer Res., 17 (6): 1264-1277 (2011)). Current treatments for MM often result in remission, but most patients eventually relapse and die (eg, Lonial et al., Supra and Rajkumar, Nature Rev. Clinical Oncol., 8 (8): See 479-491 (2011)). Although allogeneic hematopoietic stem cell transplantation has been shown to induce immune-mediated myeloma cell ablation, this approach is also highly toxic and few patients are cured (eg, Lonial et al., Supra, and Salit et al.). , Clin. Lymphoma, Myeloma, and Leukemia, 11 (3): 247-252 (2011)). Currently, there are no clinically effective FDA-approved monoclonal antibodies or autologous T cell therapies against MM (eg, Richardson et al., British J. Haematology, 154 (6): 745-754 (2011) and Yi, Cancer Journal. , 15 (6): See 502-510 (2009)).

Adoptive immune cell transfer of T cells genetically modified to recognize malignant tumor-associated antigens,
Promising new approaches to cancer treatment (eg, Morgan et al., Science, 314 (5796): 126-129 (2006); Brenner et al., Current Opinion in Immunology, 22 (2): 251-257 (eg. 2010); Rosenberg et al., Nature Reviews Cancer, 8 (4): 299-308 (2008), Kershaw et al., Nature Reviews Immunology, 5 (12): 928-940 (2005); and Pule et al., Nature Medicine, 14 ( 11): See 1264-1270 (2008)). T cells can be genetically modified to express chimeric antigen receptors (CARs), which are fusion proteins consisting of an antigen recognition moiety and a T cell activation domain (eg, Kershaw et al., Supra, Eshhar et al., Proc). . Natl. Acad. Sci. USA, 90 (2): 720-724 (1993), and Sadelain et al., Curr. Opin. Immunol., 21 (2): 215-223 (2009)).

For malignant tumors of the B cell lineage, a T cell adoptive immune cell approach utilizing anti-CD19CARs has been developed and made significant progress (eg, Jensen et al., Biology of Blood and Marrow Transplantation, 16: 1245-1256). (2010); Kochenderfer et al., Blood, 116 (20): 4099-4102 (2010); Porter et al., The New England Journal of Medicine, 365 (8): 725-733 (2011); Savoldo et al., Journal of Clinical Investigation , 121 (5): 1822-1826 (2011), Cooper et al., Blood, 101 (4): 1637-1644 (2003); Brentjens et al., Nature Medicine, 9 (3): 279-286 (2003); and Kalos Et al., Science Translational Medicine, 3 (95): 95ra73 (2011)).
Leukemia and lymphoma were cured by adopted anti-CD19CAR transduced T cells (eg, Cheadle et al., Journal of Immunology, 184 (4): 1885-1896 (2010); Brentjens.
Et al., Clinical Cancer Research, 13 (18 Pt 1): 5426-5435 (2007) and Kochenderfer et al., Blood, 116 (19): 3875-3886 (2010)). In early clinical trials, adoptive cell transfer of anti-CD19CARs transduced T cells eradicated normal and malignant B cells in patients suffering from leukemia and lymphoma (eg, Kochenderfer et al., Blood, 116 (eg, Kochenderfer et al., Blood, 116). 20): 4099-4102
(2010); Porter et al., Supra, Brentjen et al., Blood, 118 (18): 4817-4828 (2011) and Kochenderfer et al., Blood, December 8, 2011 (see publication ahead of print (2012)).
However, CD19 is rarely expressed on malignant plasma cells of multiple myeloma (eg, Gupta et al., Amer. J. Clin. Pathology, 132 (5): 728-732 (2009) and Lin. Et al., Amer. J. Clin. Pathology, 121 (4): 482-488 (2004)).

Thus, there is still a need for compositions that can be used in the treatment of multiple myeloma. The present invention provides such compositions and treatments.

[A brief summary of the invention]
The present invention comprises an isolated or purified nucleic acid sequence comprising an antigen recognition moiety and a T cell activation moiety, wherein the antigen recognition moiety encodes a chimeric antigen receptor (CAR) directed against B cell mature antigen (BCMA). I will provide a.

FIGS. 1A and 1B are graphs showing experimental data showing mode of expression of BCMA of various human cell types determined using quantitative PCR. The results are expressed as the number of BCMA cDNA copies per 10 ⁵ actin cDNA copies. In FIG. 2A-2L, the cell surface BCMA expression described in Example 1 was detected on the multiple myeloma cell line (strained cells), but not on other types of cells. It is a graph which shows the experimental data which shows. For all plots, the solid line represents staining of the anti-BCMA antibody and the dashed line represents the staining of the isotype-matched control antibody. All plots were captured on living cells. FIG. 3A is a schematic representation of a nucleic acid construct encoding anti-BCMA CAR. From the N-terminus to the C-terminus, the anti-BCMA CAR comprises an anti-BCMA scFv, a hinge and transmembrane region of the CD8α molecule, a cytoplasmic portion of the CD28 molecule and a cytoplasmic moiety of the CD3ζ molecule. FIG. 3B-3D is a graph showing experimental data showing that anti-bcma1CAR, anti-bcma2CAR and SP6CAR (described in Example 2) were expressed on the surface of T cells. Minimal anti-Fab (antigen binding fragment) staining occurred on non-transduced (UT) cells. Plots were captured on viable CD3 ⁺ lymphocytes. The number of plots indicates the percentage of cells in each quadrant. 4A-4C are graphs showing experimental data showing that T cells expressing anti-BCMA CAR degranulate T cells in a BCMA-specific manner, as described in Example 3. Plots were captured on viable CD3 ⁺ lymphocytes. The number of plots indicates the percentage of cells in each quadrant. 5A-5D are graphs showing experimental data showing that T cells expressing anti-BCMA CAR degranulate T cells in a BCMA-specific manner, as described in Example 3. Plots were captured on viable CD3 ⁺ lymphocytes. The number of plots indicates the percentage of cells in each quadrant. 6A-6C show experimental data showing that T cells expressing anti-BCMA CAR produce IFNγ, IL-2, TNF cytokines in a BCMA-specific manner, as described in Example 3. It is a graph showing. Plots were captured on viable CD3 ⁺ lymphocytes. The number of plots indicates the percentage of cells in each quadrant. FIG. 7A is a graph showing experimental data showing that T cells expressing anti-bcma2CAR proliferated in response specifically to BCMA. FIG. 6B is a graph showing experimental data showing that T cells expressing SP6 CAR did not specifically proliferate in response to BCMA. 7C and 7D show that T cells from donor A expressing anti-bcma2CAR were shown in multiple myeloma cell lines H929 (FIG. 6C) and RPMI8226 in a 4-hour cytotoxicity analysis at various effector: target cell ratios. 6D is a graph showing experimental data showing that (FIG. 6D) was specifically killed. T cells transduced with the negative control SP6 CAR produced much lower levels of cytotoxicity at all effector: target ratios. Cytotoxicity was measured twice at all effector: target ratios and the results are shown as standard error of mean +/- mean. FIG. 8A is a graph showing experimental data showing that BCMA is expressed on the surface of primary myeloma multiple myeloma cells taken from myeloma patient 3, as described in Example 5. The plot was captured on CD38 ^high CD56 ⁺ plasma cells, which accounted for 40% of bone marrow cells. In FIG. 8B, allogeneic T cells transduced with anti-bcma2CAR from donor C were co-cultured with non-genetically engineered bone marrow cells of myeloma patient 3, as described in Example 5. Later, it is a graph showing experimental data showing that IFNγ was produced. FIG. 7B also shows that T cells from the same allogeneic donor expressing anti-bcma2CAR produced much lower amounts of IFNγ when cultured with peripheral blood mononuclear cells (PBMC) from myeloma patient 3. Show that. Furthermore, T cells from donor C expressing SP6 CAR did not specifically recognize the bone marrow of myeloma patient 3. FIG. 8C shows that the plasmacytoma resected from myeloma patient 1, as shown by BCMA (solid line) and isotype-matched control staining (dashed line) fluid cell measurements (analytical method), consisted of 93% plasma cells. Is a graph showing experimental data showing that BCMA was expressed in the primary plasma cells of. FIG. 8D is a graph showing experimental data showing that T cells from myeloma patient 1 expressing anti-bcma2CAR specifically respond to autologous plasmacytoma cells to produce IFNγ. FIG. 8E is a graph showing experimental data showing that T cells from myeloma patient 1 expressing anti-bcma2CAR specifically killed autologous plasmacytoma cells at a low anti-target effector ratio. In contrast, T cells from SP6 CAR-expressing myeloma patient 1 showed low levels of cytotoxicity to autologous plasmacytoma cells. Cytotoxicity was measured twice at all effector: target ratios and the results were expressed as mean +/- standard error. FIG. 9A is a graph showing experimental data showing that anti-bcma2CAR transduced T cells can destroy multiple myeloma tumors made in mice. FIG. 9B is a graph showing the survival rate of mice with tumors treated with T cells expressing anti-bcma2CAR as opposed to controls.

[Detailed description of the invention]
The present invention provides an isolated or purified nucleic acid sequence encoding a chimeric antigen receptor (CAR), which CAR comprises an antigen recognition moiety and a T cell activation moiety. A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or poly that contains an antigen-binding domain (eg, a single chain variable fragment (scFv)) of an antibody that has bound to a T cell signaling or T cell activation domain. It is a peptide. CARs have the ability to utilize the antigen-binding properties of monoclonal antibodies to direct T cell specificity and reactivity towards selected targets, in embodiments not limited to MHC. This antigen recognition, not limited to MHC, gives T cells expressing CAR the ability to recognize antigen independently of antigen processing, thus allowing them to jump over the major mechanisms of tumor avoidance. .. In addition, CARs have the advantage of not forming dimers with endogenous T cell receptor (TCR) alpha and beta chains when expressed on T cells.

The "nucleic acid sequence" is intended to include a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and contains unnatural or altered nucleotides. The terms "nucleic acid" and "polynucleotide", as used herein, refer to nucleotides in polymer form, including any length, either ribonucleotide (RNA) or deoxyribonucleotide (DNA). These terms refer to the primary structure of a molecule and include double-stranded and single-stranded DNA as well as double-stranded and single-stranded RNA. The term also refers to nucleotide homologues such as, but not limited to, methylated and / or capped polynucleotides and homologues of either RNA or DNA obtained from modified polynucleotides. , Included as a homologue.

By "isolated" is meant the removal of nucleic acid from the natural environment. "Purified" is any of the natural sources (including genomic DNA and mRNA), synthesized under laboratory conditions (including cDNA) and / or amplified. It means that the purity of nucleic acid has been increased, where "purity" is a relative term, not "absolute purity". However, nucleic acids and proteins may be composed with diluents or adjuvants, or may be isolated for practical purposes. For example, the nucleic acid is typically mixed with an acceptable carrier or diluent when used for introduction into cells.

The nucleic acid sequence of the present invention encodes a CAR containing an antigen recognition moiety directed against a B cell mature antigen (BCMA, also known as CD269). BCMA is a member of the tumor necrosis factor receptor superfamily (eg, Thompson et al., J. Exp. Medicine, 192 (1): 129-135 (2000) and Mackay et al., Annu. Rev. Immunol., 21: See 231-264 (2003)). BCMA binds to B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) (see, eg, Mackay et al., Supra, and Kalled et al., Immunological Reviews, 204: 43-54 (2005)). Among non-malignant cells, BCMA has been reported to be mostly expressed in a subset of plasma cells and mature B cells (eg, Laabi et al., EMBO J., 11 (11): 3897-3904 (1992); Laabi et al., Nucleic Acids Res., 22 (7): 1147-1154 (1994); Kalled et al., Supra; O'Connor et al., J. Exp. Medicine, 199 (1): 91-97 (2004) and Ng Et al., J. Immunol., 173 (2): 807-817 (2004)). BCMA-deficient mice are healthy and have a normal number of B cells, but the viability of long-lived plasma cells is diminished (eg, O'Connor et al., Supra; Xu et al., Mol. Cell. Biol., 21 (12): 4067-4074 (2001); and Schiemann et al., Science, 293 (5537): 2111-2114 (2001)). BCMA RNA has been detected on the surface of plasma cells from patients with multiple myeloma by several researchers (eg, Novak et al., Blood, 103 (2): 689-694 (2004); Neri et al. , Clinical Cancer Research, 13 (19): 5903-5909 (2007); Bellucci et al., Blood, 105 (10): 3945-3950 (2005); and Moreaux et al.,
Blood, 103 (8): 3148-3157 (2004)).

The nucleic acid sequence of the present invention encodes a CAR containing an antigen-recognizing portion or an antigen-binding portion thereof containing a monoclonal antibody directed against BCMA. As used herein, the term "monoclonal antibody" means an antibody produced by a single clone of B cells that binds to the same epitope. On the other hand, a "polyclonal antibody" is a group of antibodies produced by various B cells and bound to various epitopes of the same antigen. The antigen recognition portion of CAR encoded by the nucleic acid sequence of the present invention may be an entire antibody or an antibody fragment. The entire antibody typically consists of four polypeptides, two identical copies of the heavy chain (H) polypeptide and two identical copies of the light chain (L) polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal stationary (CH1, CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one. Contains one C-terminal stationary (CL) region. Each pair of light and heavy chain variable regions forms the antigen-binding portion of the antibody. The VH and VL regions have the same general structure, and each region contains four framework regions in which the sequences are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2 and CDR3, form "hypervariable regions" involved in antigen binding.

The terms "antibody fragment,""antibodyfragment,""functional fragment of antibody," and "antigen-binding moiety" are used interchangeably herein to retain the ability to specifically bind to an antigen. Means one or more fragments or portions of an antibody to be made (see, eg, Holliger et al., Nat. Biotech, 23 (9): 1126-1129 (2005)). The antigen recognition portion of CAR encoded by the nucleic acid sequence of the present invention includes any BCMA-binding antibody fragment. The antibody fragment preferably comprises, for example, one or more CDRs, variable regions (or parts thereof), constant regions (or parts thereof) or a combination thereof. Examples of antibody fragments include: (i) Fab fragment, which is a single-chain fragment consisting of VL, VH, CL and CH1 domains; (ii) bivalent fragment containing two Fab fragments linked by dissulfide cross-linking at the hinge region. F (ab') 2 fragment; (iii) an Fv fragment consisting of a single arm VL and VH domain of an antibody; (iv) allow two domains to be synthesized as a single polypeptide chain. Single-chain Fv (scFv), a single-chain Fv (scFv) consisting of two domains of Fv fragments (ie, VL and VH) coalesced by a synthetic linker (eg, Bird et al., Science, 242: 423-426 (1988); Huston et al. , Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988) and Osbourn et al., Nat. Biotechnol., 16: 778 (1998)
(See) and (v) Bispecific antibody that is a dimer of the polypeptide chain (in the antibody, each polypeptide chain is a peptide that is too short to pair between VH and VL on the same polypeptide chain. The linker contains VH bound to VL, thereby causing pairing between complementary domains on various VH-VL polypeptide chains, producing dimer molecules with two functional antigen binding sites. ), But is not limited to these. Antibody fragments are known in the art and are described in more detail, for example, in US Patent Publication 2009/0093024 A1. In a preferred embodiment, the antigen recognition portion of the CAR encoded by the nucleic acid sequence of the invention comprises an anti-BCMA single chain Fv (scFv).

The antigen binding moiety or fragment of the monoclonal antibody may be of any size as long as the moiety binds to anti-BCMA. In this regard, the antigen binding site or fragment of the monoclonal antibody directed to BCMA (sometimes referred to herein as "anti-BCMA monoclonal antibody") is preferably about 5 to 18 (eg, about 5). , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or the range specified by any two of the above values).

In one embodiment, the nucleic acid sequence of the invention encodes an antigen recognition moiety comprising a variable region of an anti-BCMA monoclonal antibody. In this regard, the antigen recognition moiety comprises a light chain variable region, a heavy chain variable region or both a light chain variable region and a heavy chain variable region. Preferably, the antigen recognition portion of CAR encoded by the nucleic acid sequence of the present invention comprises a light chain variable region and a heavy chain variable region of an anti-BCMA monoclonal antibody. Heavy and light chain monoclonal antibody amino acid sequences that bind to BCMA are disclosed, for example, in WO 2010/104949.

In another embodiment, the nucleic acid sequence of the invention encodes a CAR containing a signal sequence. The signal sequence may be present at the amino terminus of the antigen recognition moiety (eg, the variable region of the anti-BCMA antibody). The signal sequence may contain any suitable signal sequence. In one embodiment, the signal sequence is a human granulocyte macrophage colony stimulating factor (GM-CSF) receptor sequence or a CD8α signal sequence.

In another embodiment, the CAR comprises a hinged array. Those skilled in the art will appreciate that the hinge sequence is a short sequence of amino acids that enhances the flexibility of the antibody (eg, Woof et al., Nat. Rev. Immunol., 4 (2): 89-99. (2004)). The hinge sequence is the antigen recognition part (
For example, it may be present between the anti-BCMA scFv) and the T cell activating moiety. The hinge sequence may be any suitable sequence derived from or obtained from any of the suitable molecules. For example, in one embodiment, the hinge sequence is derived from a human CD8α or CD28 molecule.

The nucleic acid sequence of the present invention encodes a CAR containing a T cell activating moiety. The T cell activation part is
Any suitable moiety derived from or obtained from any of the suitable molecules may be used.
In one embodiment, for example, the T cell activation moiety comprises a transmembrane domain. The transmembrane domain may be any of the transmembrane domains derived or obtained from any of the molecules known in the art. For example, the transmembrane domain can be obtained or derived from a CD8α molecule or a CD28 molecule. CD8 is a transmembrane glycoprotein that acts as a co-receptor for the T cell receptor (TCR) and is primarily expressed on the surface of cytotoxic (damaging) T cells. The most common form of CD8 exists as a dimer consisting of CD8α and CD8β chains. CD28 is expressed on T cells and provides the co-stimulatory signals required for T cell activation. CD28 is a receptor for CD80 (B7.1) and CD86 (B7.2). In a preferred embodiment, CD8α and CD28 are human.

In addition to the transmembrane domain, the T cell activation moiety further contains the intracellular (ie, cytoplasmic) T cell signaling domain. The intracellular T cell signaling domain is a CD28 molecule, a CD3 zeta (ζ) molecule or a modified version thereof, a human Fc receptor gamma (FcRγ) chain, a CD27 molecule, an OX40 molecule, a 4-1BB molecule or known in the art. It can be obtained or derived from other intracellular signaling molecules. As mentioned above, the CD28 molecule is an important T cell marker in T cell co-stimulation. CD3ζ works in conjunction with TCRs to generate signals and contains tyrosine-based activation motifs (ITAMs) of immune receptors. 4-1BB, also known as CD137, transmits a strong co-stimulation signal to T cells, promotes differentiation, and enhances the longevity of T lymphocytes. In a preferred embodiment, CD28, CD3 Zeta, 4-1BB, OX40 and CD27 are human.

The T cell activation domain encoded by the nucleic acid sequence of the present invention may contain a combination of any one of the above-mentioned transmembrane domains and any one or more of the above-mentioned intracellular T cell signaling domains. For example, the nucleic acid sequences of the invention can encode CARs that include the CD28 transmembrane domain and the intracellular T cell signaling domains of CD28 and CD3 zetas. Alternatively, the nucleic acid sequence of the invention encodes a CAR containing a CD8α transmembrane domain and an intracellular T cell signaling domain of CD28, CD3 zeta, Fc receptor gamma (FcRγ) chain and / or 4-1BB. Can be done.

In one embodiment, the nucleic acid sequences of the invention are 5'to 3', granulocyte macrophage colony stimulating factor (GM-CSF receptor) signal sequences, anti-BCMA scFv, hinges and transmembrane regions of human CD8α molecules. It encodes CARs containing the cytoplasmic T-cell signaling domain of the human CD28 molecule and the T-cell signaling domain of the CD3ζ molecule. In another embodiment, the nucleic acid sequences of the invention are 5'to 3', the human CD8α signal sequence, anti-BCMA scFv, the hinge and transmembrane domain of the human CD8α molecule, the cytoplasmic T cell signaling domain of the human CD28 molecule and Encode CARs containing the cytoplasmic T-cell signaling domain of the human CD3ζ molecule. In another embodiment, the nucleic acid sequences of the invention are 5'to 3', human CD8α signal sequences, anti-BCMA scFv, hinge and transmembrane regions of human CD8α molecules, cytoplasmic T cell signaling of human 4-1BB molecules. It encodes a CAR containing a domain and / or a cytoplasmic T-cell signaling domain of a human OX40 molecule and a T-cell signaling domain of a human CD3ζ molecule. For example, the nucleic acid sequence of the present invention is SEQ ID NO: 1, SEQ ID NO: 2.
Or contains or consists of a nucleic acid sequence of SEQ ID NO: 3.

The invention further provides an isolated or purified chimeric antigen receptor (CAR) encoded by the nucleic acid sequence of the invention.

The nucleic acid sequence of the present invention is of any length as long as the CAR retains biological activity, eg, the ability to specifically bind an antigen, detect diseased cells in mammals, treat or prevent disease in mammals, and the like. CARs can also be encoded, i.e., CARs that may contain any number of amino acids can be encoded. For example, the CAR is 50 or more (for example, 60 or more, 100).
More than or more than or more than 500 amino acids, but including less than 1,000 (eg, 900 or less, 800 or less, 700 or less or 600 or less) amino acids. Preferably, the CAR is about 50 to about 700 amino acids (eg, about 70, about 80, about 90, about 150, about 200, about 300, about 400, about 550 or about 650 amino acids), about 100. From about 500 amino acids (eg, about 125, about 175, about 225, about 250, about 275, about 325, about 350, about 375, about 425, about 450 or about 475 amino acids) or any of the above numbers 2 It is the range specified by the above.

Nucleic acid sequences encoding functional portions of CAR described herein are also included within the scope of the invention. When used in the context of CAR, the term "functional portion", whether it is a portion or fragment of the CAR of the invention, is the biological activity of the CAR (parent CAR) in which it is part. Refers to any part or fragment that holds. The functional portion includes, for example, a portion of CAR that retains the ability to recognize target cells and detect, treat or prevent disease to a degree similar to, to the same extent or higher than that of the parent CAR. CAR in relation to the nucleic acid sequence encoding the parent CAR
The nucleic acid sequence encoding the functional portion of is, for example, encoding a protein containing about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR. Can be done.

The nucleic acid sequence of the present invention can also encode a functional portion of CAR with amino acids added to the amino or carboxy terminus or both ends that are not found in the amino acid sequence of the parent CAR. It is desirable that the additional amino acids do not interfere with the biological function of the functional portion, such as recognition of target cells, detection of cancer, treatment or prevention of cancer. The additional amino acid is more preferably one that enhances the biological activity of CAR as opposed to the biological activity of the parent CAR.

The invention also provides nucleic acid sequences encoding the functional variants of CAR described above. When the term "functional variant" is used herein, the CAR encoded by the nucleic acid sequence of the invention.
A CAR, polypeptide or protein having substantial or significant sequence identity or similarity, meaning a functional variant that retains the biological activity of the original CAR. Functional variants include, for example, variants of the CAR (parent CAR) described herein that retain the ability to recognize target cells to a degree similar to, equal to, or higher than that of the parent CAR. .. With respect to the nucleic acid sequence encoding the parent CAR, the nucleic acid sequence encoding the functional variant of CAR is, for example, about 10% identity, about 25% identity, about 30% identity with the nucleic acid sequence encoding the parent CAR. It has about 50% identity, about 65% identity, about 80% identity, about 90% identity, about 95% identity or about 99% identity. good.

Functional variants include, for example, the amino acid sequence of CAR encoding the nucleic acid sequence of the invention, comprising at least one conservative amino acid substitution. "Conservative amino acid substitution", "conservative mutation" means that one amino acid is replaced with another amino acid having common properties. One functional way to define common properties between individual amino acids is to analyze the frequency with which amino acid exchanges between corresponding proteins in homologous organisms are normalized (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such an analysis, amino acids in the same group can predominantly exchange with each other, thus defining the group of amino acids whose effects on the overall structure of the protein are most similar to each other (). Schulz, GE and Schirmer, RH, supra). Examples of conservative mutations include, for example, substitution of lysine with arginine to maintain positive charge and vice versa; substitution of serine with threonine to maintain free OH and free NH ₂ . Amino acid substitutions of amino acids within the above subgroups such as maintenance of glutamine replacement with asparagine and the like can be mentioned.

Alternatively or in addition, the functional variant may contain the amino acid sequence of the parent CAR with at least one non-conservative amino acid substitution. "Non-conservative mutations" include amino acid substitutions between different groups, such as, for example, substitution of lysine with tryptophan, substitution of phenylalanine with serine, and the like. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions may enhance the biological activity of the functional variant so that the biological activity of the functional variant may be increased relative to the parent CAR.

The nucleic acid sequences of the invention can encode CARs containing synthetic amino acids in place of one or more natural amino acids, including functional portions and variants thereof. Such synthetic amino acids are well known in the art and are, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3 and trans-4. Hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexyl Glycin, Indolin-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N', N '-Dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α- (2-amino-2-norbornan) -carboxylic acid , Α, γ-diaminobutyric acid, α, β-diaminopropionic acid, homophenylalanine, α-tert-butylglycine and the like.

The nucleic acid sequences of the invention are glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, eg, cyclized by dissulfide cross-linking or the like. CARs that have been converted to acid additions and / or optionally dimerized, polymerized, or conjugated (including their functional parts and functional variants) can be encoded.

In a preferred embodiment, the nucleic acid sequence of the present invention comprises SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID. NO: 11 or SEQ ID NO:
Encodes a CAR containing or consisting of 12 amino acid sequences.

The nucleic acid sequence of the present invention can be prepared using a method known in the art. For example, nucleic acid sequences, polypeptides and proteins are standard recombinant DNA methods (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ^ed ., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al. , Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994). In addition, synthetically produced nucleic acid sequences encoding CAR can be isolated and / or purified from sources such as plants, bacteria, insects, mammals (eg, rats, humans, etc.). Isolation and purification methods are well known in the art. Alternatively, the nucleic acid sequences described herein can also be commercially synthesized. In this regard, the nucleic acid sequences of the invention may be synthetic, recombinant, isolated and purified.

The present invention also provides a vector containing a nucleic acid sequence encoding the CAR of the present invention. The vector may be, for example, a plasmid, cosmid, viral vector (eg, retrovirus or adenovirus) or phage. Suitable vectors and vector preparation methods are well known in the art (see, eg, Sambrook et al., Supra, and Ausubel et al., Supra).

In addition to the nucleic acid sequences of the invention encoding the CAR, the vector preferably comprises a promoter, enhancer, polyadenylation signal, transcription terminator, internal ribosome entry site (IRES), etc. that regulates the expression of the nucleic acid sequence in the host cell. Contains expression control sequences such as. Examples of expression control sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

Numerous promoters are well known in the art, including constitutive, inducible, and inhibitory promoters from a variety of sources. Representative sources of promoters include, for example, viruses, mammals, insects, plants, yeasts and bacteria, and suitable promoters from these sources are readily available or, for example, such as ATCC. It can also be made synthetically based on sequences publicly available from depository institutions and other commercial or personal sources. Promoters can be unidirectional (ie, those that initiate transcription in one direction) or bidirectional (ie, those that initiate transcription in either 3'or 5'). May be good. Examples of promoters include, but are not limited to, T7 bacterial expression system, pBAD (araA) bacterial expression system, cytomegalovirus (CMV) promoter, SV40 promoter and RSV promoter. .. Inducible promoters include, for example, the Tet system (US Pat. Nos. 5,464,758 and 5,814,618), the Ecdysone inducible system (No et al., Proc. Natl. Acad. Sci., 93: 3346-3351 (1996), T-REX). ^TM system (Invitrogen, Carlsbad, CA), LACSWITCH ^TM system (Stratagene, San Diego, CA) and Cre-ERT tamoxifen inducible recombinase system (Indra et al., Nuc. Acid. Res., 27: 4324-4327 ( 1999); Nuc. Acid. Res., 28: e99
(2000); US Pat. No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308: 123-144 (2005)). ..

As used herein, the term "enhancer" refers to, for example, a DNA sequence that enhances transcription of an operably linked nucleic acid sequence. Enhancers may be located many kilobases away from the codon region of the nucleic acid sequence and can mediate regulatory binding, DNA methylation type or DNA structural changes. Numerous enhancers from a wide variety of sources are well known in the art and as cloned polynucleotides or in their inclusion (eg, from depository institutions such as the ATCC and other commercial or personal sources). It is available. A large number of polynucleotides containing promoters (such as the commonly used CMV promoters)
It also contains an enhancer sequence. The enhancer may be present upstream, in, or downstream of the codon sequence. The term "Ig enhancer" refers to an enhancer element derived from an enhancer region located within the immunoglobulin (Ig) locus (such enhancers include, for example, heavy chain (mu) 5'enhancer, light chain. (Kappa) 5'enhancer, kappa and mu intron enhancer and 3'enhancer, etc. (generally Paul WE (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pp. 353-363. And UK Enhancer 5,885,827
See)).

The vector may also contain a "selectable marker gene". The term "selectable marker gene" is used herein in the presence of a corresponding selective agent in which cells expressing a nucleic acid sequence are selected specifically or not specifically towards it. Refers to a nucleic acid sequence that enables. Suitable selectable marker genes are well known in the art and are described, for example, in WO 1992/08796 and 1994/28143; Wigler et al., Proc.
Natl. Acad. Sci. USA, 77: 3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78: 1527 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA , 78: 2072 (1981); Colberre-Garapin et al., J. Mol. Biol., 150: 1 (1981); Santerre et al., Gene, 30: 147 (1984); Kent et al., Science, 237: 901-903 ( 1987); Wigler et al., Cell, 11: 223 (1977); Szybalska
& Szybalski, Proc. Natl. Acad. Sci. USA, 48: 2026 (1962); Lowy et al., Cell, 22: 817
(1980); and US Pat. Nos. 5,122,464 and 5,770,359.

In some embodiments, the vector can replicate in the host cell and will continue to exist as a segment of extrachromosomal DNA in the host cell in the presence of appropriate selective pressure.
"Episome expression vector" or "episome" (see, eg, Conese et al., Gene Therapy, 11: 1735-1742 (2004)). A typical commercially available episome expression vector is the Epstein-Barr nuclear antigen (see, eg, Conese et al., Gene Therapy, 11: 1735-1742 (2004)). Epstein Barr Nuclear Antigen 1) (EBNA1) and Epstein Barr Virus (EBV) Episome plasmids that utilize origin replication (oriP) include, but are not limited to, Invitrogen ( Representative of Carlsbad, CA) vectors pREP4, pCEP4, pREP7 and pcDNA3.1 and Stratagene (La Jolla, CA) pBK-CMV using T antigen and SV40-derived replication in place of EBNA1 and oriP. Examples of episomal vectors can be given, but are not limited to these.

Other suitable vectors include integrated expression vectors that include recombinant sites that randomly integrate into the host cell's DNA or allow specific recombination between the expression vector and the host cell's chromosomes. .. Such an integrated expression vector can utilize the endogenous expression control sequence of the host cell chromosome to effectively achieve the expression of the desired protein. Examples of vectors that integrate in a site-specific manner include, for example, the flp-in system of Invitrogen (Carlsbad, CA) (eg, pcDNA ^TM 5 / FRT) or Stratagene (La Jolla, CA). ) PExchange-6 Core Vectors can be mentioned as a component of the cre-lox system. Examples of vectors that randomly integrate into host cell chromosomes include, for example, Invitrogen (Carlsbad, CA) pcDNA3.1 (when introduced in the absence of T antigen) and Promega (Madison,). WI) pCI or pFN10A (ACT) FLEXI ^TM .

Viral vectors can also be used. Typical viral vectors include adenovirus-based vectors (eg, adenovirus-based Per.C6 systems available from Crucell, Inc. (Leiden, Netherlands)), lentivirus-based vectors (eg, Life Technologies (Carlsbad)). , CA) lentivirus-based pLP1))) and retrovirus vector (
Examples include, but are not limited to, Stratagene (La Jolla, CA) pFB-ERV plus pCFB-EGSH). In a preferred embodiment, the viral vector is a lentiviral vector.

The vector containing the nucleic acid sequence encoding the CAR of the present invention can be introduced into a host cell (including a suitable prokaryotic cell or eukaryotic cell) capable of expressing the CAR encoding. Preferred host cells can grow easily and reliably, have a reasonably fast growth rate, have a well-characterized expression system, and are easily and efficiently transformed or traited. It is a cell to be transferred.

As used herein, the term "host cell" refers to a cell that can contain an expression vector of any type. The host cell may be, for example, a eukaryotic cell such as a plant, animal, fungus or algae, or may be, for example, a prokaryotic cell such as a bacterium or a prokaryotic animal. The host cell may be a cultured cell or a primary cell, that is, a cell immediately isolated from an organism such as, for example, a human. The host cell may be an adhesive cell or a suspended cell, that is, a cell that grows in a suspension. Suitable host cells are well known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells and the like. To amplify or replicate the recombinant expression vector, the host cell may be, for example, DH5 (a prokaryotic cell such as a cell; to make the recombinant CAR, the host cell may be a mammalian cell. The host cell is preferably a human cell. The host cell may be of any type, of any type of tissue origin, or of any developmental stage. In one embodiment, the host cell can be a peripheral blood lymphocyte (PBL), a peripheral blood mononuclear cell (PBMC) or a natural killer (NK). The host cell is a natural killer (NK) cell. The host cell is T.
More preferably, it is a cell. Methods for selecting suitable mammalian host cells and methods for cell transformation, culture, amplification, screening and purification are well known in the art.

The present invention provides a host cell expressing the nucleic acid sequence of the present invention encoding the CAR described herein. In one embodiment, the host cell is a T cell. The T cells of the present invention may be any T cell, such as cultured T cells, for example, T cells obtained from primary T cells or cultured T cell lines, or T cells obtained from mammals. If T cells are obtained from mammals, they can be obtained from a variety of sources including, but not limited to, bone marrow, lymph nodes, thymus or other tissues or body fluids. T cells may be concentrated or purified. T cells are preferably human T cells (eg, those isolated from humans, etc.). The T cells are CD4 ⁺ / CD8 ⁺ double positive T cells, CD4 ⁺ helper T cells (eg, Th ₁ and Th ₂ cells), CD8 ⁺ T cells (eg, cytotoxic (cytotoxic) T cells), tumor invasion. cell,
It may be of any growth stage, including (but not limited to) memory T cells, naive T cells, and the like. In one embodiment, the T cells are CD8 ⁺ T cells or CD4 ⁺ T cells. T-cell lines are obtained from, for example, the American Type Culture Collection (ATCC, Manassas, VA) and the German Collection of Microorganisms and Cell Cultures (DSMZ). Possible and includes, for example, Jurkat cells (ATCC TIB-152), Sup-T1 cells (ATCC CRL-1942), RPMI 8402 cells (DSMZ ACC-290), Karpas 45 cells (DSMZ ACC-545) and derivatives thereof. Is done.

In another embodiment, the host cell is a natural killer (NK) cell. NK cells are cytotoxic (damaging) lymphocytes that play an important role in the innate immune system. NK cells are defined as large granular lymphocytes and constitute a third type of cell differentiated from the normal lymphocyte progenitor cells that produce B and T lymphocytes (eg ^{Immunobiology} , 5th ed). ., Janeway et al., Eds., Garland Publishing, New York, NY (2001)). NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils and thymus. After maturation, NK cells enter the circulation as large lymphocytes with characteristic cytotoxic granules. NK cells can recognize and kill abnormal cells such as tumor cells and virus-infected cells, and are considered to be important in innate immune defense against intracellular pathologies. As mentioned above in relation to T cells, the NK cells may be any NK cells, such as cultured NK cells, eg, NK cells from a primary NK cell or cultured NK cell line, or NK cells obtained from mammals. If NK cells are obtained from mammals, the NK cells can be obtained from a number of sources including, but not limited to, blood, bone marrow, lymph nodes, thymus or other tissues or body fluids. NK cells may be concentrated or purified. NK cells are preferably human NK cells (eg, those isolated from humans, etc.). NK cell lines are available, for example, from the American Type Culture Collection (ATCC, Manassas, VA), such as NK-92 cells (ATCC CRL-2407), NK92MI cells (ATCC). CRL-2408 and its derivatives).

The nucleic acid sequence encoding the CAR of the present invention is "transformation", "transformation" or "transduction".
Can be introduced into cells. As used herein, "transduction,""transformation," or "transduction" means the introduction of one or more foreign polynucleotides into a host cell, which is good for physical or chemical methods. .. Many transfection techniques are known in the art, for example, calcium phosphate DNA coprecipitation (eg, Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991). ); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-promoted fine particle bombing (Johnston, Nature, 346: 776-777 (1990))
) And strontium phosphate DNA coprecipitation (Brash, Mol. Cell Biol., 7: 2031-2034 (1987))
And so on. After growing the infected particles in suitable packaging cells, many of which are commercially available, phage or virus can be introduced into the host cells.

Without being bound by any particular theory or mechanism, CARs encoding the nucleic acid sequences of the invention are believed to elicit an antigen-specific response to BCMA, causing one or more of the following: BCMA expression Targeted destruction of cancer cells, reduction or elimination of cancer cells, promotion of invasion of immune cells into tumor sites, and improvement / expansion of anticancer response. Thus, the present invention contacts one or more of the isolated T cells or natural killer cells described above with a population of multiple myeloma cells expressing BCMA, thereby producing CAR and multiple myeloma. BCMA on tumor
To provide a method of destroying multiple myeloma cells, including destroying multiple myeloma by binding to. As mentioned above, multiple myeloma, also known as plasma cell myeloma or Kahler's disease, is a plasma cell cancer that is a type of white blood cell that normally plays a role in antibody production. (Raab et al., Lancet, 374: 324-329 (2009)). 1-4 people per 100,000 people get multiple myeloma per year. The disease affects more men, and for unknown reasons, it affects twice as many African Americans as White Americans. Multiple myeloma is the least common malignant hematological malignancies (14%) and accounts for 1% of all cancers (Raab et al., Supra). Treatment of multiple myeloma is typically high-dose chemotherapy followed by (allogeneic or autologous) hematopoietic stem cell transplantation, but multiple myeloma treated in this way. In patients with myeloma, a fairly high rate of relapse is common. As mentioned above, BCMA is caused to a large extent by multiple myeloma cells (see, eg, Novak et al., Supra; Neri et al., Supra; Bellucci et al., Supra; and Moreaux et al., Supra. ).

One or more isolated T cells expressing the nucleic acid sequence encoding the anti-BCMA CAR of the invention described herein are ex vivo, in vivo or in vitro. It can be contacted with a population of multiple myeloma cells expressing BCMA. “Ex vivo”
Refers to a method performed intracellularly or intracellularly in an artificial extrabiological environment with minimal changes in natural conditions. In contrast, "in vivo" refers to a method performed in an organism in a normal intact state, while "in vitro" method refers to a component of an organism released from the normal biological environment. Refers to the method performed using. The methods of the invention preferably include ex vivo and in vivo components. In this regard, for example, the isolated T cells described above can be cultured ex vivo under conditions expressing the nucleic acid sequence encoding the anti-BCMA CAR of the present invention, followed by direct multiple myeloma. It can be transplanted into affected mammals (preferably humans). Such a cell transplantation method is called "adoptive cell transfer (ACT)" in the technical field, and the transplantation passively transplants the immune-induced cells into a new recipient host to immunize the donor. The induced cell function is transferred to the new host. Adoptive cell transfer methods for the treatment of various types of cancer, including hematologic cancers such as myeloma, are known in the art, eg, Gattinoni et al., Nat. Rev. Immunol., 6 (5) :. 383-393 (2006); June, CH, J. Clin. Invest., 117 (6): 1466-76 (2007); Rapoport et al., Blood, 117 (3): 788-797 (2011) and Barber et al., It is disclosed in Gene Therapy, 18: 509-516 (2011).

The present invention also provides a method of destroying Hodgkin lymphoma cells. Hodgkin lymphoma (formerly known as Hodgkin's disease) is a cancer of the immune system revealed by the presence of a multinucleated cell type called Reed-Sternberg cells. The two major types of Hodgkin lymphoma include classical Hodgkin lymphoma and nodular lymphocyte-dominant Hodgkin lymphoma. Hodgkin lymphoma is currently treated with radiation therapy, chemotherapy, and hematopoietic stem cell transplantation, with treatment options selected according to the patient's age, sex, stage of progression, size, and histological subtype of disease. BCMA expression is detected on the surface of Hodgkin lymphoma cells (see, eg, Chiu et al., Blood, 109 (2): 729-739 (2007)).

When T cells or NK cells are administered to mammals, the cells may be allogeneic or autologous. In a "self-derived" method of administration, cells (eg, hematopoietic stem cells or lymphocytes) are removed from the mammal, stored (modified if necessary) and returned to the same mammal. In the "allogeneic" method of administration, mammals receive cells (eg, hematopoietic stem cells or lymphocytes) from donors that are genetically similar but not identical. The cells are preferably autologous cells of the mammal.

T cells or NK cells are preferably administered to humans in the form of compositions such as, for example, pharmaceutical compositions. Alternatively, the CAR-encoding nucleic acid sequence of the present invention or a vector containing a CAR-encoding nucleic acid sequence can be formulated into a composition such as a pharmaceutical composition and then administered to humans. The pharmaceutical composition of the present invention can include a population of T cells or NK cells expressing the CAR of the present invention. The pharmaceutical composition is added to the host cell expressing the nucleic acid sequence of the present invention or the CAR of the present invention.
Contains other pharmaceutically active agents and medications such as chemotherapeutic agents such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. May be. In a preferred embodiment, the pharmaceutical composition comprises a population of isolated T cells or NK cells expressing the CAR of the invention, more preferably T cells or NK cells expressing the CAR of the invention.

The T cells or NK cells of the invention can be provided in the form of salts, for example, pharmaceutically acceptable salts. Suitable pharmaceutically acceptable acid addition salts include, for example, hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphate, inorganic acids such as nitrate and sulfuric acid and tartaric acid, acetic acid, citric acid, malic acid, lactic acid, etc. Examples thereof include salts derived from organic acids such as fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, arylsulfonic acid such as p-toluenesulfonic acid and the like.

When selecting a carrier, one is determined by the host cell expressing the specific nucleic acid sequence, vector, CAR of the present invention, and the host cell expressing the nucleic acid sequence, vector, CAR of the present invention is administered. It is also determined by the specific method used for. Therefore, there are various suitable compositions of the pharmaceutical composition of the present invention. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives include, for example, methylparaben, propylparaben, sodium benzoate salt, benzalconium chloride and the like. Two or more kinds of preservatives may be used at will. The preservative or mixture thereof typically comprises from about 0.0001% to about 2% by weight of the total composition.

Further, a buffer may be used in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphate, potassium phosphate and various other acids and salts. Two or more kinds of buffering agents may be used arbitrarily. The buffer or mixtures thereof typically contain from about 0.001% to about 4% by weight of the total composition.

Methods of preparing administrable (eg, parenteral) compositions are well known to those of skill in the art, and further, for example, Remington: The Science and Practice of Pharmacy, LippincottWilliams &Wilkins; 21st ed. It is described in detail on May 1, 2005).

The composition containing the nucleic acid sequence encoding the CAR of the present invention or the host cell expressing the CAR can be formed, for example, as an inclusion body such as a cyclodextrin inclusion body or as a liposome. Liposomes help target host cells (eg, T cells or NK cells) or nucleic acid sequences of the invention to specific tissues. Liposomes can also be utilized to extend the half-life of the nucleic acid sequences of the invention. For the production of liposomes, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980) and US Pat. No. 4,235,871, 4,501,728, 4,837,
Many methods can be used, including the methods described in 028 and 5,019,369.

The compositions are sustained release, delayed and sustained release agents so that the compositions of the invention can be reached prior to or with sufficient time to sense the treatment site. The system can be adopted. Various forms of emission arrival systems are available and are well known to those of skill in the art. Such a system is particularly suitable for some of the composition embodiments of the present invention, as it avoids repeated administration of the composition, thereby increasing convenience for the patient and the physician.

The composition comprises a host cell expressing the nucleic acid sequence encoding the CAR of the present invention or a vector containing the nucleic acid sequence of the present invention in an amount effective for treating or preventing multiple myeloma or Hodgkin lymphoma. Is desirable. In the present application, terms such as "treatment" and "treating" mean obtaining the desired pharmacological and / or physiological effects. The effect is preferably therapeutic, i.e., the effect of partially or completely treating the disease and / or the undesired symptoms caused by the disease. To this end, the method of the invention comprises a "therapeutically effective amount" of a host cell expressing the nucleic acid sequence encoding the CAR of the invention or a vector comprising the nucleic acid sequence of the invention. Includes administration of. "Therapeutically effective amount" means the dose required to achieve the desired therapeutic result and the effective amount for the required period. The therapeutically effective amount may vary depending on the patient's individual disease status, age, sex and weight and the ability of CAR to elicit the desired responsiveness in the patient. For example, a therapeutically effective amount of the invention is an amount that binds to and destroys BCMA on multiple myeloma cells.

Alternatively, the pharmacological and / or physiological effect is a prophylactic, i.e., an effect that completely or partially prevents the disease or condition. In this regard, the method of the invention comprises a "preventively effective amount" of a composition comprising a host cell expressing the nucleic acid sequence encoding the CAR of the invention or a vector comprising the nucleic acid sequence of the invention, multiple myeloma. Includes administration to mammals with a predisposition to swelling or Hodgkin lymphoma. "Prophylactically effective amount" means the dose required to achieve the desired prophylactic outcome (eg, prevention of disease onset, etc.) and the effective amount for the required period.

Typical amounts of host cells administered to mammals (eg, humans) are, for example, cells in the range of about 1 million to about 100 billion, but amounts below or beyond this exemplary range are also in the invention. It belongs to the range of. For example, the daily dose of host cells of the invention is from about 1 million to about 50 billion cells (eg, about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells. , About 20 billion cells, about 30 billion cells, about 40 billion cells or the range defined by any two of the above numbers), preferably about 10 million to about 100 billion cells (eg, about 2000). 10,000 cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 750 Billion cells, about 90 billion cells, or the range defined by any two of the above numbers), more preferably about 100 million to about 50 billion cells (eg, about 120 million cells, about 250 million). Cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, or any of the above figures. The range defined by two numerical values).

Therapeutic or prophylactic efficacy can be monitored by periodically assessing the patient receiving treatment. Depending on the condition, repeated administration may be performed over a period of several days or longer, in which case the treatment is repeated until the disease symptoms are suppressed to a desired degree. However, other dosing therapies may also be useful and they also fall within the scope of the invention. As a preferred method of administration, the composition may be administered in a single high-dose instantaneous dose, or the composition may be administered in a continuous infusion.

Compositions comprising a host cell expressing a nucleic acid sequence encoding the CAR of the present invention or a vector containing the nucleic acid sequence encoding the CAR of the present invention may be oral, intravenous, intraperitoneal, subcutaneous, transpulmonary, transpulmonary. It can be administered to mammals using standard dosing techniques including cutaneous, intramuscular, nasal, buccal tablets, sublingual tablets or suppository administration. Preferably, the composition is suitable for parenteral administration. The term "parenteral" includes, in the present application, intravenous, intramuscular, subcutaneous, rectal, transvaginal and intraperitoneal administration. The composition is more preferably administered to mammals using peripheral systemic delivery by intravenous, intraperitoneal or subcutaneous injection.

A composition comprising a host cell expressing a nucleic acid sequence encoding the CAR of the present invention or a vector containing the nucleic acid sequence encoding the CAR of the present invention may be administered with one or more additional therapeutic agents that can be administered in combination with mammals. May be good. “Combination” means that in addition to a composition comprising a host cell of the invention or a vector of the invention, one or more additional therapeutic agents can enhance the effect of one or more additional therapeutic agents. Alternatively, it means administration within a sufficiently close time so that the effect of the composition of the present invention can be enhanced. In this regard, the host cell of the invention or the composition comprising the vector of the invention may be administered first, followed by one or more additional therapeutic agents, and vice versa. .. Alternatively, a composition comprising the host cells of the invention or the vector of the invention can be administered simultaneously with one or more additional therapeutic agents. An example of a therapeutic agent that can be used in combination with the host cell of the present invention or the composition containing the vector of the present invention is IL-2.

Once a composition comprising a host cell expressing a nucleic acid sequence encoding the CAR of the present invention or a vector containing the nucleic acid sequence encoding the CAR of the present invention is administered to a mammal (eg, human), the biological activity of the CAR is increased. , Can be measured by any suitable method known in the art. According to the method of the invention, CAR binds to BCMA on multiple myeloma cells and the multiple myeloma cells are destroyed. Binding of CAR to BCMA on the surface of multiple myeloma cells can be analyzed using suitable methods well known in the art such as, for example, ELIZA and fluid cell analysis. The ability of CAR to destroy multiple myeloma cells is described, for example, in Kochenderfer et al., J. Immunotherapy, 32 (7): 689-702 (2009) and Herman et al., J. Immunological Methods, 285 (1): 25-40. It can be measured using any suitable method well known in the art, such as the cytotoxicity analysis described in (2004). The biological activity of CAR can also be measured by analyzing the expression of cytokines such as CD107a, IFNγ, IL-2 and TNF.

Those skilled in the art will readily appreciate that the nucleic acid sequences encoding CARs of the invention can be modified in a variety of ways such that the therapeutic or prophylactic efficacy of CARs can be enhanced by modification. There will be. For example, CAR can be conjugated directly to the targeting moiety or indirectly via a linker. Methods of conjugating a compound, eg, CAR, to a targeted moiety are known in the art. See, for example, Wadwa et al., J. Drug Targeting 3: 111 (1995) and US Pat. No. 5,087,616.

The invention will be shown more specifically by the following examples, but it goes without saying that these examples should not be construed as limiting the scope of the invention in any way.

Example 1
This example demonstrates the expression pattern of BCMA in human cells.

Quantification using BCMA-specific primers and probe sets (Life Technologies, Carlsbad, CA) on a panel of cDNA samples from a wide range of normal tissues contained in human major tissue qPCR panel II (Origine Technologies, Rockville, MD). A real-time polymerase chain reaction (qPCR) was performed. The cDNA derived from the plasmacytoma cells resected from a patient with advanced multiple myeloma was analyzed as a positive control. RNA, RNeasy mini kit (Qiagen, Inc., Valencia, CA)
Was extracted from plasmacytoma cells and cDNA was synthesized using standard methods. BCMA cDNA (Origine Technologies, Rockville, MD) A standard curve for BCMA qPCR was generated by diluting the full-length plasmid in carrier DNA. qPCR accurately detected the number of copies from 10 ² to 10 ⁹ BCMA copies per reaction. The number of β-actin cDNA copies in the same tissue was quantified using the Taqman β-actin primer and probe kit (Life Technologies, Carlsbad, CA). The β-actin standard curve was created by amplifying the continuous dilution of the β-actin plasmid. All qPCR reactions were performed on a Roche LightCycler 480 instrument (Roche Applied Sciences, Indianapolis, IN).

The results of qPCR analysis are shown in FIGS. 1A and 1B. As measured by fluid cell measurement, 93% of the cells from the plasmacytoma sample were plasma cells. BCMA expression in plasmacytoma samples was significantly higher than in other tissues. BCMA cDNA was detected in several blood tissues such as peripheral blood mononuclear cells (PBMC), bone marrow, spleen, lymph nodes and tonsils. Low levels of BCMA cDNA were detected in most gastrointestinal organs, such as the duodenum, rectum and stomach. Expression of BCMA in the gastrointestinal organs may be the result of the presence of plasma cells and B cells in the gut-associated lymphoid tissues such as lamina propria and Peyer's patches (eg, Brandtzaeg, Immunological Investigations, 39 (4-5): 303). -See 355 (2010)). Low levels of BCMA cDNA are also detected in the testes and trachea. The low levels of BCMA cDNA detected in the trachea may be due to the presence of plasma cells in the lamina propria of the trachea. (See, for example, Soutar, Thorax, 31 (2): 158-166 (1976)).

Cell surface BCMA expression of various cell types is expressed in multiple myeloma cell lines H929, U266 and RPMI8226.
Including, further characterized using fluid cell measurements (see Figure 2A-2L). Multiple myeloma Cell lines H929, U266 and RPMI8226 all expressed cell surface BCMA. In contrast to this
Sarcoma cell line TC71, T cell leukemia line CCRF-CEM and kidney cell line 293T-17 did not express cell surface BCMA. Primary CD34 ⁺ hematopoietic cells, primary airway epithelial cells, primary bronchial epithelial cells and primary intestinal epithelial cells all had no cell surface BCMA expression.

The results of this example show that BCMA is expressed on the surface of multiple myeloma cells and exhibits a limited expression pattern in normal tissues.

Example 2
This example describes the construction of nucleic acid sequences encoding the anti-BCMA chimeric antigen receptors (CARs) of the present invention.

Two mouse anti-human BCMA antibodies, named "C12A3.2" and "C11D5.3", were obtained from International Patent Application Publication Publication WO2010/104949 (Kalled et al.). The amino acid sequences of the heavy and light chain variable regions of these antibodies were used to design single chain variable fragments (scFvs) with the following general structure:
Light chain variable region-linker-heavy chain variable region.

The linker has the following amino acid sequence:
GSTSGSGKPGSGEGSTKG (SEQ ID NO: 7) (eg Cooper et al., Blood, 101 (4): 1637-1644
See (2003).

We designed DNA sequences encoding two chimeric antigen receptors, each containing the following elements in 5'to 3':
CD8α signal sequence, anti-BCMA scFv above, hinge and transmembrane domain of human CD8α molecule, CD28
The cytoplasmic portion of the molecule and the cytoplasmic portion of the CD3ζ molecule.
A schematic diagram of the nucleic acid sequence encoding CAR is shown in FIG. 3A. CARs containing variable regions from C12A3.2 and C11D5.3 were named anti-bcma1 and anti-bcma2, respectively.

The anti-bcma2 CAR described above, each with a different signal sequence and T cell activation domain.
We designed a DNA sequence encoding five additional chimeric antigen receptors based on. In this regard, the 8ss-anti-bcma2 CAR contained the following elements in 5'to 3':
CD8α signal sequence, scFv, hinge and transmembrane domain of human CD8α molecule, cytoplasmic portion of CD28 molecule and cytoplasmic moiety of CD3ζ molecule.
G-Anti-bcma2 CAR contained the following elements in 5'to 3':
Human GM-CSF receptor signal sequence, scFv, hinge and transmembrane domain of human CD8α molecule, cytoplasmic portion of CD28 molecule and cytoplasmic moiety of CD3ζ molecule.
Anti-bcma2-BB CAR contained the following elements from 5'to 3':
CD8α signal sequence, scFv, hinge and transmembrane domain of human CD8α molecule, cytoplasmic portion of 4-1BB molecule and cytoplasmic moiety of CD3ζ molecule.
Anti-bcma2-OX40 CAR contained the following elements from 5'to 3':
CD8α signal sequence, scFv, hinge and transmembrane region of human CD8α molecule, cytoplasmic moiety of OX40 molecule (see, eg, Latza et al., European Journal of Immunology, 24: 677-683 (1994)) and cytoplasmic moiety of CD3ζ molecule.
Anti-bcma2-BBOX40 contained the following elements from 5'to 3':
CD8α signal sequence, scFv, hinge and transmembrane region of human CD8α molecule, cytoplasmic portion of 4-1BB molecule, cytoplasmic moiety of OX40 molecule and cytoplasmic moiety of CD3ζ molecule.
Table 1 shows the elements present in each of the seven CAR arrays.

The sequences used for CD8α, CD28, CD3 Zeta, 4-1BB (CD137) and OX40 (CD134) were obtained from the database of the publicly available National Center for Biotechnology Information (NCBI).

The nucleic acid sequence encoding CAR is, for example, Kochenderfer et al., J. Immunology, 32 (7): 689-7.
It was prepared using a method well known in the art as described in 02 (2009) and Zhao et al., J. Immunology, 183 (9): 5563-5574 (2009). The nucleic acid sequence encoding each CAR was a codon optimized and synthesized using GeneArt ^TM technology (Life Technologies, Carlsbad, CA) at the appropriate restriction site.

The sequences encoding anti-bcma1 and anti-bcma2 CARs were ligated into a lentiviral vector plasmid named pRRLSIN.cPPT.MSCV.coDMF5.oPRE (eg, Yang et al., J. Immunotherapy, 33 (6) :. See 648-658 (2010)). The coDMF5 portion of this vector was replaced with the CAR-encoding nucleic acid sequence using standard methods. The resulting anti-BCMA CAR vectors were named pRRLSIN.cPPT.MSCV. Anti-bcma1.oPRE and pRRLSIN.cPPT.MSCV. Anti-bcma2.oPRE. We also constructed a negative control CAR containing SP6 scFv that recognizes haptens 2,4,6-trinitrophenyl (eg Gross et al., Proc. Natl. Acad. Sci. USA, 86 (24): 10024-10028 (1989). reference). This CAR was called SP6. SP6 CAR was cloned into the same lentiviral vector as anti-BCMA CARs. It contained the same signaling domains as anti-bcma1 and anti-bcma2. A supernatant containing the lentivirus encoding each CAR was prepared by the protocol described in Yang et al., Supra. Specifically, the following plasmids were transfected into 293T-17 cells (ATCC CRL-11268): pMDG (encoding the vesicular stomatitis virus envelope protein), pMDLg / pRRE (encoding the HIV Gag and Pol proteins). ), PRSV-Rev (encoding the RSV Rev protein) and plasmids encoding anti-bcma CARs (see, eg, Yang et al., Supra).

For example, using standard methods such as those described in Hughes et al., Human Gene Therapy, 16: 457-472 (2005), G-anti-bcma2, 8ss-anti-bcma2, anti-bcma2-BB. , Anti-bcma2-OX40 and anti-bcma2-BBOX40 CARs-encoding sequences were ligated to obtain a gammaretrovirus vector plasmid called MSGV (mouse stem cell virus-based splice-gag vector). After creating the gammaretrovirus plasmid that encodes CAR, a retrovirus with an RD114 envelope that lacks replication ability is described in Kochenderfer et al., J. Immunotherapy, 32 (7): 689-702 (2009). 293-Prepared by transient transfection of base packaging cells.

Human T cells were transduced using the lentivirus lacking replication ability and the retrovirus encoding the above CARs. For anti-bcma1 and anti-bcma2, T cells were cultured as described above (
See, for example, Kochenderfer et al., J. Immunotherapy, 32 (7): 689-702 (2009)), 5% Human AB
Anti-CD3 monoclonal antibody OKT3 (Ortho-Biotech, Horsham, PA) and 300 international units (IU) / in AIM ^VTM medium (Life Technologies, Carlsbad, CA) containing serum (Valley Biomedical, Winchester, VA). Stimulation was given by mL of interleukin-2 (Novartis Diagnostics, Emeryville, CA). Thirty-six hours after the start of culture, activated T cells were suspended in protamine sulfate and a lentivirus supernatant containing 300 IU / mL IL-2. The cells were centrifuged at 1200 xg for 1 hour. Then, T cells were cultured at 37 ° C. for 3 hours. The supernatant was then diluted 1: 1 with RPMI medium (Mediatech, Inc., Manassas, VA) + 10% fetal bovine serum (Life Technologies, Carlsbad, CA) and IL-2. T cells were cultured overnight in this diluted supernatant and then returned to IL-2-added AIM ^VTM medium (Life Technologies, Carlsbad, CA) + 5% human AB serum culture. T cells were stained with biotin-labeled polyclonal goat anti-mouse-F (ab) ₂ antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) to detect anti-BCMA CARs. Anti-bcma1 CAR, anti-bcma2 CAR on transduced T cells, as shown in Figure 3B-3D.
And high levels of cell surface expression of SP6 CAR were observed.

For G-anti-bcma2, 8ss-anti-bcma2, anti-bcma2-BB, anti-bcma2-OX40 and anti-bcma2-BBOX40 CARs, peripheral blood mononuclear cells at a rate of 1x10 ⁶ cells per mL, Suspended in T cell medium containing 50 ng / mL anti-CD3 monoclonal antibody OKT3 (Ortho, Bridgewater, NJ) and 300 IU / mL IL-2. Phosphate buffered saline at a concentration of 11 μg / mL of ^{RETRONECTIN TM} polypeptide (Takara Bio, Shiga, Japan), a recombinant polypeptide of the human fibronectin fragment that binds viruses to cell surface proteins. Dissolved in (PBS) solution, 2 mL of PBS solution of RETRONECTIN ^TM polypeptide was added to each well of a non-tissue culture coated 6-well plate (BD Biosciences, Franklin Lakes, New Jersey). The plates were incubated at room temperature (RT) for 2 hours. After incubation, the RETRONECTIN ^TM solution was aspirated and 2 mL of a blocking solution consisting of Hanks balanced salt solution (HBSS) plus 2% bovine serum albumin (BSA) was added to each RETRONECTIN ^TM -coatwell. The plates were incubated at room temperature (RT) for 30 minutes. The blocking solution was sucked out and the wells were washed with a solution of HBSS + 2.5% (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid) (HEPES). The retrovirus supernatant was quickly liquefied, diluted 1: 1 in T cell medium, and then 2 mL of diluted supernatant was added to each RETRONECTIN ^TM -Coatwell. After adding the supernatant, the plates were centrifuged at 2000 xg for 2 hours at 32 ° C. Then, the supernatant was sucked out from the wells, and 2x10 ⁶ T cells previously cultured with OKT3 antibody and IL-2 for 2 days were added to each well. When adding T cells to a retrovirus coated plate, T cells were suspended in T cell medium containing 300 IU / mL IL-2 at a concentration of 0.5x10 ⁶ cells per mL. After adding T cells to each well, the plates were centrifuged at 1000xg for 10 minutes. The plates were incubated overnight at 37 ° C. Transduction was repeated the next day. After 18-24 hours of incubation, T cells are removed from the plate and suspended in fresh T cell medium containing 300 IU / mL IL-2 at a concentration of 0.5x10 ⁶ cells per mL, 5% at 37 ° C. Cultured with CO ₂ . High levels of cell surface expression of anti-bcma2-BBOX40, anti-bcma2-BB and 8ss-anti-bcma2 were observed on transduced T cells.

The results of this example demonstrate a method for producing a nucleic acid sequence encoding CAR of the present invention and a method for expressing CAR on the surface of T cells.

Example 3
This example describes a series of experiments used to determine the specificity of the CAR of the present invention for BCMA.

cell
NCI-H929, U266 and RPMI8226 are all BCMA + multiple myeloma cell lines obtained from ATCC (ATCC Nos. CRL-9068, TIB-196 and CCL-155, respectively). A549 (ATCC No. CCL-185)
Is a BCMA-negative lung cancer cell line. TC71 is a BCMA-negative sarcoma cell line. CCRF-CEM is a BCMA-negative T cell line (ATCC No. CCL-119). BCMA-K562 is a K562 cell (ATCC No. CCL-243) transduced with a nucleic acid sequence encoding the full length of BCMA. NGFR-K562 is a K562 cell transduced with a gene encoding a low-affinity nerve growth factor (see, eg, Kochenderfer et al., J. Immunotherapy., 32 (7): 689-702 (2009)). Suffering from multiple myeloma 3
Peripheral blood lymphocytes (PBLs) from human patients (ie, myeloma patients 1-3) were used, but those PBLs were derived from three other subjects, donor A, donor B, and donor C. It is PBL. All donors A to C had melanoma. CD34 + primary cells are from 3 normal and healthy donors. Plasma cell tumor samples were obtained from myeloma patient 1 and bone marrow samples were obtained from myeloma patient 3. All of the above human samples were taken from patients enrolled in IRB-approved clinical trials at the National Cancer Institute. Primary human epithelial cells of small airway epithelial cells, bronchial epithelial cells and intestinal epithelial cells were obtained from Lonza, Inc. (Basel, Switzerl and).

Interferon-Gamma and TNF ELIZA
BCMA-positive or BCMA-negative cells are placed in two wells of 96-well round bottom plates (Corning Life Sciences, Lowell, MA) in AIM ^VTM medium (Life Technologies, Carlsbad, CA) + 5% human serum. Combined with CAR transduced T cells. Plates were incubated at 37 ° C for 18-20 hours. After incubation, ELISA for IFNγ and TNF was performed using standard methods (Pierce, Rockford, IL).

As shown in Table 2 (all units are pg / mL of IFNγ), T cells transduced with anti-bcma1 or anti-bcma2 CARs were cultured overnight with BCMA-expressing cell line BCMA-K562. They produced large amounts of IFNγ, but when cultured with the negative control cell line NGFR-K562, CAR transduced T cells only produced background levels of IFNγ.

* Effector cells are T cells from a patient with multiple myeloma (myeloma patient 2). The T cells are transduced or non-transduced with the CAR shown above.
** The indicated target cells were incubated overnight with effector cells and subjected to IFNγELISA.

T cells expressing 8ss-anti-bcma2, anti-bcma2-BB and anti-bcma2-OX40 CARs are T cells and targets as shown in Table 3 (all units are pg / mL of IFNγ). When the cells were co-cultured overnight, they produced IFNγ in a specific response to BCMA + target cells.

T cells transduced with anti-BCMA CARs produced large amounts of IFNγ when co-cultured overnight with BCMA-expressing multiple myeloma cell lines. In contrast, anti-BCMA CARs produced much lower amounts of IFNγ when cultured with various BCMA-negative cell lines. In contrast to T cells transduced with anti-bcma1 CAR, anti-bcma2 CAR and their variants (ie, 8ss-anti-bcma2, anti-bcma2-BB and anti-bcma2-OX40) are transduced. The resulting T cells produced more IFNγ when cultured with BCMA-positive cells and smaller amounts of IFNγ when cultured with BCMA-negative cells.

T cells transduced with the anti-bcma2 CAR variant were subjected to overnight T cells and target cells as shown in Table 4 (all units are pg / mL of tumor necrosis factor (TNF)). When co-cultured, BCMA +
TNF was produced in response specifically to target cells.

T cells transduced with anti-bcma2 CAR and its variants are T cells transduced with anti-bcma1 CAR.
Since BCMA-expressing cells were recognized slightly stronger and more specifically than cells, only anti-bcma2CAR and its variants were used in the following experiments.

CD107a Analysis Two T cell populations were prepared in two separate tubes. One tube contained BCMA-K562 cells and the other tube contained NGFR-K562 cells. In addition, T cells transduced with anti-bcma2 CAR and anti-bcma2 CAR variants in both tubes, 1 mL of AIM ^VTM medium (Life Technologies, Carlsbad, CA) + 5% human serum, a given concentration of anti-. CD107a antibody (eBioscience, Inc., San Diego, CA; clone eBioH4A3) and 1 μL Golgi Stop (BD Biosciences, Franklin Lakes, NJ) were added. All tubes were incubated at 37 ° C. for 4 hours and then stained for expression of CD3, CD4 and CD8.

CAR transduced T cells from three different subjects increased CD107a expression in a specific response to stimulation by BCMA-expressing target cells (see Figure 4A-4C). This indicates that BCMA-specific T cell degranulation, a requirement for perforin-mediated cytotoxicity, has occurred (eg, Rubio et al., Nature Medicine, 9 (11): 1377-1382 (2003)). reference). In addition, T cells expressing the anti-bcma2 CAR mutant 8ss-anti-bcma2, anti-bcma2-BB, and anti-bcma2-OX40 are shown in Figure 5A-5D.
As shown in, when stimulated with target cells in vitro, they were degranulated in a BCMA-specific manner.

Intracellular Cytokine Staining Analysis (ICCS)
Populations of BCMA-K562 cells and populations of NGFR-K562 cells were prepared in two separate tubes as described above. T cells transduced with anti-bcma2 CAR from 2 myeloma patients in both tubes, 1 mL AIM V medium (Life Technologies, Carlsbad, CA) + 5% human serum and 1 μL Golgi Stop ( BD Biosciences, Franklin Lakes, NJ) was added. All tubes were incubated at 37 ° C for 6 hours. Cells were surface stained with anti-CD3, anti-CD4 and anti-CD8 antibodies. Cells were permeabilized and treated with IFNγ (BD Biosciences, Franklin Lakes, NJ, clone B27), IL-2 (BD Biosciences, Franklin Lakes, NJ, clone MQ1-17H12) and TNF (BD Biosciences, Franklin Lakes, NJ, clones). For MAb11), intracellular staining was performed according to the instructions of Cytofix / Cytoperm kit (BD Biosciences, Franklin Lakes, NJ).

Much of the population of anti-bcma2 CAR transduced T cells from myeloma patient 2 was stimulated with BCMA-expressing target cells for 6 hours, as shown in Figure 6A-6C, followed by BCMA-specific cytokine IFNγ. , IL-2 and TNF were produced.

Proliferation analysis We analyzed the proliferative capacity of anti-bcma2 CAR transduced T cells when stimulated with BCMA expression target cells. Specifically, BCMA-K562 cells irradiated with 0.5x10 ⁶ or NGFR-K562 cells irradiated with 0.5x10 ⁶ were transduced with either anti-bcma2 CAR or SP6 CAR for a total of 1x10 ⁶ Co-cultured with T cells. The T cells were described in Mannering et al., J. Immunological Methods, 283 (1-2): 173-183 (2003), carboxyfluorescein diacetate succin-imidazole ester (CFSE) (Life Technologies, Carlsbad, Labeled with CA). AIM ^VTM medium (Life Technologies, Carlsbad, CA) + 5% human AB serum medium was used for co-culture. IL-2 was not added to the medium. Four days after the start, live cells in each co-culture were counted using tripan blue to eliminate dead cells. This was followed by polyclonal biotin-labeled goat-anti-human BCMA antibody (R & D Systems, Minneapolis, MN), followed by streptavidin (BD Biosciences, Franklin Lakes, NJ), anti-CD38 antibody (eBioscience, Inc., San). Diego, CA)
Flow cell analysis was performed by staining T cells with anti-CD56 antibody (BD Biosciences, Franklin Lakes, NJ). Analysis of flow cell measurement data was performed using FlowJo software (Tree Star, Inc., Ashland, OR). ..

T cells expressing anti-bcma2 CAR dilute CFSE significantly more when cultured with BCMA-K562 cells than when cultured with negative control NGFR-K562 cells, as shown in Figure 7A. bottom. These results indicate that anti-bcma2 CAR transduced T cells specifically proliferate when stimulated with BCMA expression target cells. In contrast, when SP6 CAR-expressing T cells were cultured with BCMA-K562 target cells or NGFR-K562 target cells, there was no significant difference in CFSE dilution (see Figure 7B). This indicates that there is no BCMA-specific proliferation by SP6 CAR expressing T cells.

For proliferation analysis, 0.8x10 ⁶ T cells expressing anti-bcma2 CAR were initially cultured with either BCMA-K562 cells or NGFR-K562 cells. After 4 days of culture, 2.7x10 ⁶ anti-bcma2 CAR-expressing T cells were present in the BCMA-K562 cell-containing culture medium, while 0.6x10 ⁶ in the NGFR-K562 cell-containing culture medium. Only anti-bcma2 CAR-expressing T cells were present. This BCMA-specific increase in the absolute number of anti-bcma2 CAR-expressing T cells indicates that these T cells proliferated in response to BCMA.

The results of this example demonstrate that the CAR-expressing T cells of the invention exhibit BCMA-specific cytokine production, degranulation and proliferation.

Example 4
In this example, the anti-BCMA CAR expressing T cells of the present invention illustrate that they can disrupt multiple myeloma cell lines.

To determine if the anti-bcma2 CAR transduced T cells described in Examples 2 and 3 can disrupt the BCMA-expressing multiple myeloma (MM) cell line, cytotoxicity. An analysis was performed. Specifically, the cytotoxicity of target cells is described, for example, in Kochenderfer et al., J. Immunotherapy, 32 (7): 689-702 (2009) and Hermans et al., J. Immunological Methods, 285 (1): 25-40 ( It was measured by comparing the viability of BCMA expression target cells (ie, multiple myeloma cell lines H929 and RPMI8226) to the viability of negative control CCRF-CEM cells using the analytical method described in 2004). ..

Approximately 50,000 BCMA expression target cells and approximately 50,000 CCRF-CEM cells were combined in the same tube containing various numbers of CAR transduced T cells. CCRF-CEM negative control cells were labeled with 5- (and -6)-(((4-chloromethyl) benzoyl) amino) tetramethylrhodamine (CMTMR) (Life Technologies, Carlsbad, CA) fluorescent dye and expressed BCMA. Target cells were labeled with CFSE.
In all experiments, the cytotoxicity of anti-bcma2 CAR transduced effector T cells was compared to the cytotoxicity of SP6 CAR transduced negative control effector T cells obtained from the same subject. Co-culture was performed twice in sterile 5 mL tubes (BD Biosciences, Franklin Lakes, NJ) with T cell: target cell ratios of 20.0: 1, 7: 1, 2: 1 and 0.7: 1. The culture was incubated at 37 ° C for 4 hours. Immediately after incubation, 7-amino-actinomycin D (7AAD; BD Biosciences, Franklin Lakes, NJ) was added. Surviving BCMA-expressing target cells and surviving CCRF-CEM negative control cells were determined in T cell / target cell co-cultures, respectively.

For each T cell / target cell co-culture, the survival percentage of BCMA expression target cells relative to CCRF-CEM negative control cells was determined by dividing the BCMA expression target cell percentage by the CCRF-CEM negative control cell percentage. The corrected survival percentage of BCMA expression target cells is the survival percentage of BCMA expression target cells in each T cell / target co-culture medium, including only BCMA expression target cells and CCRF-CEM negative control cells but not effector T cells. Calculated by dividing by the ratio of BCMA expression target cell percentages to CCRF-CEM negative control cell percentages in vitro. This correction is necessary to account for changes due to cell number at initiation and spontaneous target cell death. Cytotoxicity is calculated as follows:
Percentage of BCMA-expressing target cells Cytotoxicity = 100-Corrected survival percentage of BCMA-expressing target cells.

The results of cytotoxicity analysis are shown in Figures 7C and 7D. T cells transduced with anti-bcma2 CAR specifically killed BCMA-expressing multiple myeloma cell lines H929 and RPMI8226. In contrast, SP6 CAR transduced T cells showed much lower levels of cytotoxicity for these cell lines.

The results of this example demonstrate that the nucleic acid sequence encoding the anti-BCMA CAR of the present invention can be used in the multiple myeloma cell line disruption method.

Example 5
This example illustrates that T cells expressing the anti-BCMA CAR of the present invention can destroy primary multiple myeloma cells.

The primary multiple myeloma cells described in Example 2 were evaluated for BCMA expression and BCMA-specific cytokine production, degranulation and proliferation using the methods described above.

Cell surface BCMA expression was detected on primary myeloma cells from myeloma patient 3 as well as on 4 primary multiple myeloma samples (see Figure 8A). BCMA-expressing plasma cells accounted for 40% of the cells of the bone marrow sample from myeloma patient 3. As shown in FIG. 8B, allogeneic T cells transduced with anti-bcma2 CAR from donor C were co-cultured with non-genetically engineered bone marrow cells from myeloma patient 3 and then IFNγ. Produced. Anti-bcma2 CAR transduced T cells from the same allogeneic donor produced much smaller amounts of IFNγ when cultured with peripheral blood mononuclear cells (PBMC) from myeloma patient 3. In addition, SP6-CAR-transduced T cells from donor C
The bone marrow of myeloma patient 3 was not specifically recognized. It has been previously reported that normal PBMCs do not contain cells expressing BCMA (see, eg, Ng et al., J. Immunology, 173 (2): 807-817 (2004)). To confirm this observation, patient 3 PBMCs were evaluated for BCMA expression by fluid cell analysis. Patient 3 PBMCs did not contain BCMA-expressing cells, apart from a small population of CD56 + CD38 ^high cells, which accounted for approximately 0.75% of PBMCs. This population is probably considered to consist of circulating multiple myeloma cells.

Plasma cells resected from myeloma patient 1 consisted of 93% plasma cells, and these primary plasma cells expressed BCMA, as shown in FIG. 8C. T cells from myeloma patient 2 produced IFNγ when cultured with non-genetically engineered plasmacytoma cells from allogeneic origin of myeloma patient 1. T cells from myeloma patient 2 did not produce significant amounts of IFNγ when cultured with PBMCs from myeloma patient 1. T cells from SP6 CAR transduced myeloma patient 2 did not produce significant amounts of IFNγ when cultured with plasmacytoma cells from myeloma patient 1 or PBMC. As a result of measurement by the fluid cell measurement method, PBMC of bone marrow patient 1 did not express BCMA.

T cells in myeloma patient 1 who received 8 cycles of prior myeloma treatment were successfully cultured and introduced with a lentiviral vector encoding anti-bcma2 CAR. Eight days after the start of culture, expression of anti-bcma2 CAR was confirmed on 65% of T cells. T cells from myeloma patient 1 expressing anti-bcma2 CAR produced IFNγ in a specific response to autologous plasmacytoma cells (Fig. 8D). T cells from myeloma patient 1 expressing SP6 CAR did not recognize autologous plasmacytoma cells. Anti-bcma2 CAR-expressing T cells and SP6 CAR-expressing T cells did not recognize autologous PBMC. In addition, T cells from myeloma patient 1 expressing anti-bcma2 CAR have an effector ratio to a low target.
Autologous plasmacytoma cells were specifically killed. In contrast, patients with myeloma expressing SP6 CAR 1
T cells from from showed only low levels of cytotoxicity to autologous plasmacytoma cells (Fig. 8E).

The results of this example demonstrate that the anti-BCMA CAR of the present invention can be used as a method for destroying primary multiple myeloma cells.

Example 6
This example illustrates that T cells expressing the anti-BCMA CARs of the present invention can destroy tumors that have been established in mice.

Immunity-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, Jackson Laboratory) were injected intradermally with 8x10 ⁶ RPMI8226 cells. Tumors were grown for 17 to 19 days, after which mice were intravenously infused with 8x10 ⁶ anti-bcma2 CAR or SP6 CAR transduced T cells. Tumors were measured with calipers every 3 days. The tumor size (area) was obtained in mm ² by multiplying the longest length and the length in the direction perpendicular to the longest length direction. The mouse was sacrificed when the longest length reached 15 mm. Animal studies were conducted with the approval of the National Cancer Institute Animal Care and Use Committee.

The results of this example are shown in FIGS. 9A and 9B. Mice treated with anti-bcma2 transduced T cells around day 6 showed tumor cell shrinkage and disappeared around day 15. In addition, all mice treated with anti-bcma2 transduced T cells survived up to 30 days after T cell infusion.

The results of this example illustrate that the anti-BCMA CAR of the present invention is capable of destroying multiple myeloma cells in vivo.

All references cited in this application, including publications, patent applications and patents, are as if each of those references were individually and specifically described and described as a whole in this application so that they may be incorporated into this application by reference. As such, to the same extent, those references are incorporated herein by reference.

In the context of describing the invention (particularly in relation to claims described below), the articles "a", "an" and "the" and similar demonstratives are used unless otherwise noted in the present application. Or it should be interpreted as covering both the singular and the plural, unless there is a clear contextual conflict. The terms "comprising", "having", "including" and "containing" are open-ended terms (ie, unless otherwise noted). , "Contains, but is not limited to,"). The description of the range of numbers given in this document is merely intended to serve as an abbreviation for individual reference to individual numbers within that range, unless otherwise stated in this document. Therefore, individual numerical values are included in this book as if they were described individually in this book. All of the methods described in this document may be carried out in any suitable order, unless otherwise noted or clearly contradictory in context. When any and all examples or exemplary words (eg, "like") are used herein, they are intended to facilitate the understanding of the invention unless otherwise noted. It is not intended to limit the scope of the invention. No language in this specification should be construed as indicating an unclaimed element as essential to the practice of the invention.

Preferred embodiments of the present invention are described herein, including the best embodiments that the inventor knows to carry out the invention. Changes in those preferred embodiments may also be apparent to those skilled in the art upon reading the above description. The inventor expects one of ordinary skill in the art to make appropriate modifications and also intends that the invention will be practiced in aspects other than those specifically described herein. Accordingly, the invention includes, to the extent permitted by applicable law, all modifications and equivalents of the subject matter described in the claims attached herein. Furthermore, any combination of the above elements, including any possible changes, is included in the invention unless otherwise noted or clearly contradictory in context.

Claims (18)

An isolated or purified nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR containing an antigen recognition moiety and a T cell activation moiety, the antigen recognition moiety being a B cell mature antigen. Oriented to (BCMA). The isolated or purified nucleic acid sequence according to claim 1, wherein the antigen recognition moiety comprises a monoclonal antibody directed against BCMA or an antigen binding moiety thereof. The isolated or purified nucleic acid sequence of claim 2, wherein the antigen recognition moiety comprises a variable region of a monoclonal antibody directed against BCMA. The isolated or purified nucleic acid sequence according to any one of claims 1-3, wherein the T cell activating moiety comprises the T cell signal domain of any of the following proteins: human CD8-alpha. Protein, human CD28 protein, human CD3 zator protein, human FcRγ protein, CD27 protein, OX40 protein, human 4-1BB protein, any of the above modifications or combinations of any of the above. The isolated or purified nucleic acid sequence according to any one of claims 1-4, wherein the nucleic acid sequence is a nucleic acid of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. Contains sequences. An isolated or purified CAR encoded by the nucleic acid sequence according to any one of claims 1-5. SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12 amino acids The isolated or purified CAR according to claim 6, which comprises a sequence. The vector comprising the isolated or purified nucleic acid sequence according to any one of claims 1-5. An isolated host cell expressing the nucleic acid sequence according to any one of claims 1-5. SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12 amino acids Claim 9 expressing CAR comprising a sequence
The isolated host cell of the description. .. The isolated host cell according to claim 9 or 10, which is a T cell. The isolated host cell according to claim 9 or 10, which is a natural killer (NK) cell. A method for disrupting multiple myeloma cells comprising contacting BCMA-expressing multiple myeloma cells with the isolated or purified nucleic acid sequence according to any one of claims 1-5. Then, CAR is produced by this method, binds to BCMA on multiple myeloma cells, and the multiple myeloma cells are destroyed. A method for disrupting multiple myeloma cells comprising contacting BCMA-expressing multiple myeloma cells with the isolated or purified vector according to claim 8, wherein the method comprises CAR.
Is produced and binds to BCMA on multiple myeloma cells, destroying multiple myeloma cells. A method for destroying multiple myeloma cells, which comprises contacting BCMA-expressing multiple myeloma cells with one or more isolated T cells according to claim 11, wherein the method produces CAR. It is produced and binds to BCMA on multiple myeloma cells, destroying the multiple myeloma cells. A method for destroying multiple myeloma cells, which comprises contacting BCMA-expressing multiple myeloma cells with one or more isolated NK cells according to claim 12, wherein the method produces CAR. It is produced and binds to BCMA on multiple myeloma cells, destroying the multiple myeloma cells. The method according to any one of claims 13-16, wherein the multiple myeloma cells are present in humans. The method according to any one of claims 13-16, wherein the multiple myeloma cells are in vitro.

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